三种冰片调控脑缺血模型TRPV1联动Notch通路重塑NVU表达性效机制的研究

The nature-effect framework of “pungent cool properties -clearing heat for resuscitation” of the three kinds of borneol involves in anti-infection, reduce cerebral infraction and regulate blood-brain barrier (BBB) based on our previous projects. Stork is a critical severe disease, the damage has a multi-stage cascade effect. In recent years, the protection of the neurovascular unit (NVU) has been regarded as a new therapeutic target. The gated channel of vanillic acid subtype 1 (TRPV1 related to pungent and temperature) that distributed in vascular endothelial cells, neurons and astrocytes(Ast) are opend after cerebral ischemia, increasing intracellular calcium influx. Calcium overload in Ast further damages the blood-brain barrier and neurons. Once cerebral ischemia occurs, Notch signaling pathway can promote vascular endothelial cell regeneration and induce Ast to transform and repair neurons. This puts forward the hypothesis that “pungent cool properties -clearing heat for resuscitation” mechanism of bronel by remolding NVU through regulating TRPV1 Channel Linkage Notch Signaling. In our research, combine with Chinese Pharmacology - Neurobiochemistry - Molecular Imaging, adopt advanced technologies such as two-photon imaging, laser confocal, and molecular docking, we study the does-time-effective relationship and remold NVU through regulating TRPV1 Channel Linkage Notch Signaling related proteins repair mechanisms of three kinds of borneol penetrating BBB of cerebral ischemia model. It reveals the scientific connotation in the nature-effect of “pungent cool properties -clearing heat for resuscitation”. There is a scientific sense of application value and innovation.

受基金资助：三种冰片具“辛凉-清热开窍”性效构架，有抗感染、降低脑梗死率，调节血脑屏障（BBB）等作用。脑卒中是危急重症，损伤呈多环节级联效应，近年将保护神经血管单元（NVU）视为治疗新靶点。脑缺血后分布于血管内皮细胞、神经元、星形胶质细胞（Ast）的香草酸亚型1（TRPV1与辛味、温度相关）通道打开，细胞钙内流增加；星形胶质细胞钙超载又损及血脑屏障及神经元。缺血时Notch信号通路可促血管内皮细胞新生、诱导Ast转化修复神经元。提出三种冰片入BBB 调控TRPV1通道联动Notch信号通路重塑NVU是表达“辛凉-清开”性效机制假说。融合中药学-神经生化学-分子影像学等知识，采用双光子成像、高内涵成像、转录组学等技术，研究三种冰片透过脑缺血模型BBB的量-时-效及调控TRPV1通道与Notch信号通路相关蛋白重塑NVU修复机制，揭示“辛凉-清开”性效整体科学内涵，有科学意义应用价值和创新。

本研究基于中医整体观思路，提出三种冰片入血脑屏障(BBB)调控TRPV1通道联动Notch信号通路重塑神经血管单元(NVU)是表达“辛凉-清开”性效假说。融合多学科知识，采用成像技术、转录组学、慢病毒转染星形胶质细胞等前沿技术，从多层次研究三种冰片改善脑缺血大鼠各脑区的病理损伤，保护BBB、调控TRPV1通道与Notch信号通路，稳定NVU的生物学机制。. 研究提示：①三种冰片能降低永久性局灶性脑缺血(pMCAO)模型大鼠体温，促进神经功能恢复，降低脑梗死率，改善皮质、海马等各脑区的病理性损伤，可能是其表达“辛微寒/凉-清开”发挥开窍醒神功效的药效学基础，强弱依次呈艾片＞天然冰片＞合成冰片趋势；可通过抑制Notch信号通路，促进损伤脑皮质部位的血管网络重建和神经发生，抗神经元凋亡，保护脑微血管和BBB，稳定和重塑NVU发挥脑神经保护作用。②三种冰片能改善脑缺血再灌注损伤(t-MCAO)急性期模型大鼠的神经功能，降低脑梗死率，抑制炎症反应从而改善缺血侧皮层、纹状体、海马等脑区的病理损伤，增加皮层脑血流，增加缺血侧不同脑区神经元尼氏体数量及NeuN表达，抑制神经元CaMKII表达以保护神经元；激活TRPV1离子通道保护脑血管神经，激活p38MAPK信号通路，下调Caspase3和Bax蛋白表达、上调Bcl-2蛋白以抑制细胞凋亡；保护BBB的完整性，稳定NVU，体现“辛-开（开窍醒神）”性效特征。③三种冰片通过促进脑缺血再灌注恢复期（t-MCAO）纹状体区星形胶质细胞转分化为神经元及神经干细胞和不同脑区的神经发生，增加皮层血流灌注改善脑梗死，抑制脑萎缩及肢体运动功能障碍，与调节Wnt及Notch通路有关。. 创新特色：①首次基于两种脑缺血模型大鼠,比较三种冰片对不同脑区、脑缺血各期的脑神经保护作用，探明其调控Notch信号通路和TRPV1离子门控通道机制，阐明“辛-开”性效科学内涵，艾片在脑神经保护及调控机制方面具有优势。②首次采用慢病毒转染技术揭示了艾片有促脑缺血恢复期纹状体区星形胶质细胞转分化为神经元、天然冰片促转分化为神经干细胞趋势，明确其对不同脑区神经发生的调控机制，为冰片治疗缺血性脑卒中恢复期提供了参考。. 成果：发表学术文章30篇，国内外学术交流5次；培养研究10人（博士6，硕士4），达到目标，并有延伸拓展。